Iris Publishers- Open access Journal of Agriculture and Soil Science | Antimicrobial Effects of Herb Extracts Against Foodborne Pathogen Listeria monocytogenes in Vitro

 

Authored by Hua Yang*,

Abstract

Herb extract is a type of well-known natural antimicrobial from plants. Food Drug Administration recognized that most herb extracts as Generally Recognized as Safe for human consumption. The objective of this study is to conduct three experiments and to evaluate the inhibitory and bactericidal effects of nine herb extracts against five representative strains of Listeria monocytogenes in vitro. In the experiment 1, each of herb extracts 2, 4, 5, 8 exhibited inhibitory effects against five strains of L. monocytogenes individually at 37 °C in Mueller-Hinton broth (MHB). The MIC values of those four herb extracts ranged between 5 - 50 mg/mL. In experiment 2, herb extract 4, which showed the lowest MIC value (5 mg/mL), reduced populations of L. monocytogenes in a range of 0.38 - 0.91 log CFU/mL after 30 min treatment at 37oC in MHB, indicating that herb extract 4 may not expected to be used as an antimicrobial agent for the purpose of reducing L. monocytogenes within a short period of time. In experiment 3, at concentrations of 1.56 and 0.78 mg/mL, herb extracts 2, 4, 5, 8 inhibited the growth of a five-strain L. monocytogenes cocktail individually at the abused refrigerator temperature of 12 oC, except herb extract 8 at the concentration of 0.78 mg/mL. At a concentration of 3.13 mg/mL, those four herb extracts reduce cell populations in a range of 2.2 to 1.6 mg/mL at 11 days. Herb extracts 2, 4, 5 and 8 could be potentially developed into food preservatives for controlling foodborne L. monocytogenes.

Keywords: Antimicrobial effect; Herb extracts; Listeria monocytogenes

Abbreviations: Herb extract (HE); Colony forming unit (CFU); Minimum inhibitory concentration (MIC); Mueller-Hinton broth (MHB); Ready-to-eat (RTE); Transfers in tryptic soy broth (TSB); Tryptic soy agar (TSA); Buffered peptone water (BPW); Phosphate-buffered saline (PBS)

Introduction

Listeria monocytogenes is a gram-positive foodborne pathogen that is widely distributed during food preparation, storage, and distribution. A variety of ready-to-eat (RTE) foods such as milk, cheeses, ice cream, raw meat, fresh vegetable and fruits may be contaminated with Listeria monocytogenes [1,2]. Consumption of foods contaminated with L. monocytogenes is linked to an increased risk of listeriosis. To control L. monocytogenes in food products, meat industry uses chemical preservatives such as sodium acetate, sodium lactate and various nitrites. However, it is acknowledged that uses of chemical antimicrobials have increased the consumer concerns and created a demand for “natural” and “minimally processed” food. As a result, there has been a great interest in natural antimicrobial agents.

Plant-derived extracts have been used since ancient times, especially in China [3] and India [4,5]. In addition to the uses as flavoring material, plant extracts and essential oils represent a natural alternative in the nutritional, pharmaceutical, and agricultural fields. Due to their antimicrobial properties, plant extracts have been suggested to be used as antioxidant and preservatives in food products, to incorporate into food packaging materials, plant and crop protectants against insect pests, and medicinal products for human and livestock [6]. In recent times, plant extracts have gained great interests especially in food industry. Most plant extracts are classified as generally recognized as safe by U.S Food and Drug Administration, and are easily degradable in human body [7,8]. Previous studies have been proven that many spices and plant essential oils exhibited inhibition and/or bactericidal effects against L. monocytogenes in food products. For example, cinnamon essential oil and oregano reduced the growth rate of L. monocytogenes by 10% and 19% respectively in ham at 4 °C [9]. Thyme and clove essential oils reduced populations of L. monocytogenes in zero-fat beef hotdogs by 1.3 log CFU/g and 1.0 log CFU/g respectively with 5 min treatment at room temperature (21 °C) [10]. The objective of this study is to evaluate potential inhibitory and bactericidal effects of nine herb extracts (HEs) against foodborne pathogenic L. monocytogenes in vitro in order to select natural antimicrobial agents for the control of foodborne L. monocytogenes in food products.

Introduction

Experimental design

HEs can be used to inhibit the growth of foodborne pathogen and/or reduce pathogen populations. In this study, we conducted three experiments to evaluate their potential uses as antimicrobial agents in food products. In experiment 1 (Exp. 1), a minimum inhibitory concentration (MIC) study was conducted to compare inhibitory effects of each of nine HEs against each of five L. monocytogenes strains individually in Mueller-Hinton broth (MHB). In experiment 2 (Exp. 2), the HE with the lowest MIC was used to determine its reductions of each of five L. monocytogenes strains individually at 37 °C for 30 min in MHB. In experiment 3 (Exp. 3), those HEs which could inhibit the L. monocytogenes growth in Exp. 1 were evaluated for their inhibitory effects against a five-strain L. monocytogenes cocktail in MHB up to 11 days at 12 °C.

Bacterial strains and growth conditions

Table 1: Bacterial strains used in the study [15].

Five strains of L. monocytogenes which isolated from epidemics were used in this study and are listed in Table 1. According to [11], these five L. monocytogenes strains were selected from a total of 46 strains which represented a genetic diversity of ribotypes, pulsedfield gel electrophoresis types, serotypes, and lineages. In addition, these five strains are believed to cover the genetic diversity of human disease- associated L. monocytogenes and to provide a valuable tool for evaluating the effectiveness of antimicrobials to inactivate or inhibit L. monocytogenes. Therefore, we used these five genetically distinct strains of L. monocytogenes to evaluate inhibitory and bactericidal efficacies of nine HEs. All strains were activated from 20% glycerol frozen stocks (-80 °C) by two transfers in tryptic soy broth (TSB) (Difco, Spark, MD) at 37 °C for 24 h and were subsequently subculture on tryptic soy agar (TSA) (Difco, Spark, MD) at 37 °C for 24h. Each activated strain was kept on TSA plates at 4 °C.

Herb extracts preparation

A total of nine types of herbs were obtained in the form of powder. Each of nine herbs was extracted with sterile deionized water followed by the procedure of [12] with modification. The HEs were prepared before the day of experiment. Each of the HEs was made by combining 10g of each herb powder with 90 mL of sterile deionized water, incubating in a water bath at 45 °C for 30 minutes, and then boiling for 15 minutes. Each of the nine HEs was then cooled to room temperature and was centrifuged at 6000 x g for 15 minutes at room temperature (Thermo Scientific Sorvall Legend X1R Centrifuge, Am Kalkberg, Germany). The supernatant of each HE was transferred into a 50 mL polypropylene tube and stored at 4 °C until use next day.

Exp. 1: Determining MICs of the HEs

Each strain of the L. monocytogenes listed in Table 1 was inoculated in TSB individually and was incubated at 37 °C for 24h. After the incubation, each strain was serially diluted in MHB (Difco, Spark, MD) to approximately 106 CFU/mL. Nine HEs were diluted with the sterile deionized water to six concentration levels: 100, 60, 30, 15, 10, 5 mg/mL. Five mL of each diluted strain was mixed with 5 mL of each diluted HEs in glass sterile test tubes, to make the final concentrations to be 50, 30, 15, 7.5, 5, 2.5 mg/mL for each HEs and approximately 5 x 105 CFU/mL for each strain. Negative control samples were prepared by combining 5mL of each of nine diluted HEs with 5 mL of MHB separately to make the same final herb concentrations for each HE listed above but without inoculum. Positive control samples were prepared by combining 5mL of each diluted strain with 5mL of MHB separately to make same final bacteria concentrations for each diluted strain listed above but without any HE. All tubes were subsequently incubated at 37 °C for 24h. After 24h incubation, all treatment and control samples were visually examined. The lowest herb concentration at which each treatment sample did not show turbidity were designated as the MIC. All tests were performed in two independent replication trails with three samples on each trail (n=6).

Exp. 2: Reduction of L. monocytogenes cells treated with the HE 4

The HE 4 exhibited inhibitory effect against L. monocytogenes with the lowest MIC in Exp. 1. In this experiment, the HE 4 was determined for its reduction of L. monocytogenes cells. After the 24h incubation, each strain was serially diluted in MHB to approximate concentration of 106 CFU/mL. The HE 4 was diluted in sterile deionized water to the concentration of 50 mg/mL. Two mL of each of diluted L. monocytogenes strains was combined with 2mL of the diluted the HE 4 separately, to make a final concentration of 25 mg/mL of the HE and approximate 5 x 105 CFU/mL of each strain. The positive control samples were prepared by combining 2mL of sterile deionized water and 2mL of each of the five diluted strains separately, to make the same concentrations of each strain as the treatment samples but without HE 4. All treatment and control samples were incubated for 30 min at 37 °C. Our preliminary data showed that the HE 4 exhibited the best reductions against each of five L. monocytogenes strains at 37 °C (data not shown). In a previous published study, thyme and clover have been reported to reduce populations of L. monocytogenes after 5 min treatment in peptone water at room temperature (21 °C) [10]. In our study, each of five strains were treated 30 min with HE 4, which was six times longer than [10]. After 30 min treatment, all treatment samples were immediately diluted with sterile deionized water to a concentration of 0.25 mg/mL for the HE 4, in order to terminate its further antimicrobial activity. Our preliminary study has shown at the concentration of 0.25 mg/mL, the HE 4 could not inhibit the growth of each of five L. monocytogenes strains (data now shown). Each of treatment and positive control samples was subsequently serially diluted in 0.1% buffered peptone water (BPW) and each diluted sample were then plated onto tryptic soy agar (TSA) with two duplications. The TSA plates were then incubated for 48 h at 37oC to enumerate the numbers of surviving L. monocytogenes cells. All tests were performed in two independent replication trails with two samples on each trail (n=4).

Exp. 3: Antimicrobial effects of HEs against L. monocytogenes cocktail at abused refrigerated temperature

In Exp. 1, HEs 2, 4, 5 and 8 which inhibited L. monocytogenes growth at or below 50 mg/mL concentrations. In this experiment, those four HEs were evaluated for their inhibitory effects at 12 °C, which represents the abused refrigeration temperature. Each of the five L. monocytogenes strains listed in Table 1 was cultured in TSB separately for 24h at 37 °C. A five-strain L. monocytogenes cocktail was prepared prior to the study. A 10-mL volume of each 24h grown culture was pooled and mixed in a 50 mL sterile falcon tube. After centrifugation at 6000 x g for 15 min at 4 °C, the supernatant was removed. The cell pellet was washed once with a 10-mL volume of phosphate-buffered saline (PBS), and subsequently resuspended in 50 mL PBS. The L. monocytogenes cocktail was serially diluted in MHB to an approximate 5 x 102 CFU/mL concentration.

The HEs 2, 4, 5, and 8 were diluted in sterile deionized water to three levels of concentrations, 6.25, 3.13 and 1.56 mg/mL. Concentrations of HEs were determined based on the preliminary data (data not shown). The treatment samples were prepared by combining 2 mL of each of four diluted HEs and 2mL of the diluted L. monocytogenes cocktail separately in glass test tubes, to make the final concentrations of each of the four HEs at three levels, 3.13, 1.56 and 0.78 mg/mL, and approximately 2.5 x 102 CFU/mL of the L. monocytogenes cocktail. The positive control samples were prepared by combining 2 mL of diluted L. monocytogenes cocktail and 2 mL of sterile deionized water separately but without any HE. Surviving cells from control samples were enumerated immediately after inoculation (day 0). All treatment and control samples were incubated for up to 11 days at 12 °C. All samples were serially diluted in 0.1% BPW and subsequently plated onto two duplicate TSA plates daily from day 1 to day 5, and every two days from day 7 to day 11. TSA plates were incubated for 48h at 37 °C to enumerate the numbers of surviving L. monocytogenes cells. Each treatment sample and control sample were performed in three independent replication trails with two samples on each trail (n=6).

Statistical Analysis

Microbiological data were converted to log CFU/mL prior to the statistical analysis. Statistical analyses were conducted using analysis of variance via the glimmix procedure of SAS (SAS Studio Basic Edition 3.8, SAS Institute, Inc., Cary, N.C.). Least square means were calculated and significant differences between means were detected at the P < 0.05 in the Exp. 2 and at P < 0.001 in the Exp. 3.

Results and Discussion

MICs of nine herb extracts

MIC is defined as the lowest concentration of an antimicrobial agent which prevents visible microbial growth under designed conditions [13]. In this study, the visible microbial growth was determined by comparing the turbidity between treatment samples and control samples after 24h incubation at 37 °C. The MIC value for each of the nine HEs against each strain are shown in Table 2. Four HEs 2, 4, 5 and 8 inhibited the growth of the five L. monocytogenes strains at MIC values ranging from 5 to 50 mg/mL. The other five HEs 1, 3, 6, 7, 9 did not exhibited inhibition effects at up to 50 mg/ mL. Based on the MIC values, the inhibitory effects of those four HEs were ranked from the strongest to weakest as follows: HE 4 (5 mg/mL) > HE 5 (15 mg/mL) > HE 2 (15-30 mg/mL) > HE 8 (50 mg/mL).

Table 2: Minimum inhibitory concentration of the nine herb extracts against five L. monocytogenes strains (n=6).

The sensitivity to different natural antimicrobials varies in some Gram-positive and Gram-negative bacteria. For example, studies have shown that Gram-positive L. monocytogenes were more sensitive to some essential oils and HEs than Gram-negative E. coli and Salmonella enterica Enteritidis [14-16]; The Ocimum sanctum extract was found to be equally effective against Gramnegative bacteria (E. coli, S. typhimurium and P. aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) [17]; however, Gram-negative pathogens, V. parahaemolyticus and S. typhimurium, were more sensitive to eugenol than Gram-positive S. aureus [18]. As a result of Exp. 1, four out of nine HEs inhibited the growth of L. monocytogenes. Further studies can be conducted to evaluate and compare the antimicrobial effects of those nine HEs against other foodborne Gram- positive and Gram-negative pathogens.

Reduction of L. monocytogenes cells treated with HE 4

In Exp. 2, the HE 4 was chosen to evaluate its reductions of five L. monocytogenes strains individually at 37 °C for 30 min treatment since HE 4 exhibited the strongest inhibition effect with the lowest MIC (5 mg/mL) in Exp. 1. After 30 min incubation with HE 4 at a concentration of 25 mg/mL, differences (P < 0.05) of surviving cells between treatment samples and control samples were observed for each of five L. monocytogenes strains (Figure 1). Cell reductions of HE 4 against five L. monocytogenes strains were calculated: N1-227 (0.91 log CFU/mL), C1-056 (0.87 log CFU/mL), R2-499 (0.85 log CFU/mL), J1-177 (0.59 log CFU/mL), N3-013 (0.38 log CFU/mL).

In a previous published study, at the concentrations of 0.5 mL/L, essential oils of thyme and clover have been reported to reduce populations of L. monocytogenes from 7.2 to 1.8 log CFU/mL and from 7.1 to 1.2 log CFU/mL respectively after 5 min treatment in peptone water at room temperature (21 °C) [10]. In addition, another study indicated that essential oil of origanum reduced populations of each of five L. monocytogenes strains in a range of 1-2 log CFU/mL after 30 min treatment in 0.9% saline solution at room temperature [19]. In our study, Although HE 4 reduced less than 1 log CFU/mL for each strain, populations of surviving cells of each strain were significant (P < 0.05) after HE4 treatment compared with control samples. The result indicated that using HE 4 solely against L. monocytogenes might be less effective than essential oils of thyme, clover and organum. However, there has been increased interests to the use natural antimicrobial agents in their combinations for controlling foodborne pathogens. The effects of the combined substances were observed to be greater than the sum of individual effects against L. monocytogenes in combinations of carvacrol/linalool [20] and oregano/rosemary [21]. HE 4 was expected to be used in combination with other compounds to increase antimicrobial effects.

Inhibitory effects and reductions of four herb extracts against L. monocytogenes cocktail at abused refrigerated temperature

Since HE 2, 4, 5 and 8 exhibited inhibitory effects against L. monocytogenes at 37 °C in Exp. 1, we expected that those four HEs could inhibit L. monocytogenes growth at 12 °C, which represented to the abused refrigerator temperature. We investigated the antimicrobial effects of HEs 2, 4, 5 and 8 at three concentration levels (3.13, 1.56, 0.78 mg/mL) against a five-strain L. monocytogenes cocktail. The initial populations of L. monocytogenes cocktail in control and all treatment samples were 2.3 log CFU/mL. For control samples without any HE, bacteria population rapidly increased from 2.3 log CFU/mL (day 0) to 8.4 log CFU/mL by 4 days, and then increased to 8.8 log CFU/mL by day 7. After 7 days, bacteria population did not have further increase in number. For treatment samples, the growth of L. monocytogenes during refrigerated storage was dependent on the type of herb and HE concentration. In general, compared with control samples, lower bacteria populations (P < 0.001) were observed in all treatments except for the HE 8 at the concentration of 0.78 mg/mL (Tables 3-5).

At a concentration of 3.13 mg/mL (Table 3), HEs 2, 4, 5 and 8 reduced inoculated L. monocytogenes populations from 2.3 log CFU/mL to 0.2, 0.1, 0.7 and 0.5 log CFU/mL at day 11, respectively. Compared with positive control samples without any HE, each of four HEs had lower bacterial population (P < 0.001) on each day from day 1 to day 11. This result indicated that at the concentration of 3.13 mg/mL, all four HEs effectively reduced bacteria populations of L. monocytogenes cocktail at 12 °C.

Table 3: Least square means ± standard deviation of Listeria monocytogenes cocktail populations in inoculated Mueller-Hinton broth with each of four herb extracts at concentration of 3.13 mg/mL or deionized water (control), stored at 12 °C for 11 days (n=6).

Table 4: Least square means ± standard deviation of Listeria monocytogenes cocktail populations in inoculated Mueller-Hinton broth with each of four herb extracts at concentration of 1.56 mg/mL or deionized water (control), stored at 12 °C for 11 days (n=6).

Table 5: Least square means ± standard deviation of Listeria monocytogenes cocktail populations in inoculated Mueller Hinton broth with each of four herb extracts at concentration of 0.78 mg/mL or deionized water (control), stored at 12 °C for 11 days (n=6).

At the concentration of 1.56 mg/mL (Table 4), HE 2 reduced L. monocytogenes populations from 2.3 log CFU/mL to 0.2 log CFU/ mL at day 11, which was 8.6 log CFU/mL lower (P < 0.001) than the control. Although counts of L. monocytogenes in the sample with HE 5 increased from 2.3 log CFU/mL to 4.0 log CFU/mL at day 11, it was still 4.8 log CFU/mL lower (P < 0.001) than the control. However, compared with the control, HE 4 and 8 were lower (P < 0.001) in bacteria populations only up to 5 days. Therefore, antimicrobial effects of those four HE at concentration of 1.56 mg/ mL were ranked from the strongest to weakest as follows: HE 2 > HE 5 > HE 4 = HE 8.

Table 5 shows the inhibitory effects of each of the four HEs in MHB at the concentration of 0.78 mg/mL. Counts of the L. monocytogenes cocktail in the sample with HE 8 were not different (P > 0.001) from the control sample on each day from day 1 to day 11, indicating that HE 8 at a concentration of 0.78 mg/mL could not inhibit bacterial growth. Counts of samples with HE 4 or 5 increased from 2.3 log CFU/ mL to 6.7 and 6.4 log CFU/mL by day 4 respectively, which were lower (P < 0.001) than the control by about 2 log CFU/mL. After 4 days of incubation, the bacterial population of the sample with HE 4 were not different (P > 0.001) with the positive control sample on each day from day 5 to day 11. Although the sample with HE 5 did not show different (P > 0.001) in bacteria population with the positive control sample at day 5 and day 7, the population of HE 5 was 0.5 log CFU/mL lower (P < 0.001) than the control at day 9 and day 11. In addition, comparing with positive control samples, HE 2 slowed the microbial growth and reached to 5.6 log CFU/mL by day 5, which was lower than the controls for 3 log CFU/mL (P < 0.001). After 7 days of incubation, the bacterial population of the sample with HE 2 were not different (P > 0.001) with the control sample on each day from day 7 to day 11. In summary, at the concentrations of 0.78 mg/mL, HE 2 inhibited the microbial growth up to 5 days; HE 4 and 5 inhibited L. monocytogenes growth up to 4 days; HE 8 could not inhibit the microbial growth.

The demand for convenience foods such as RTE foods has increased in recent years. The majority of listeriosis cases are foodborne [22] and linked to the consumption of RTE foods which are contaminated with L. monocytogenes. Due to the high mortality rate of listeriosis, the U.S. Department of Agriculture and the FDA labels L. monocytogenes as an adulterant of RTE foods. Examples of RTE foods that support the growth of L. monocytogenes are milk, high fat dairy products, soft unripened cheese, cooked and raw seafood, deli-type salads, sandwiches, fresh-cut vegetable and fruits [23] and the processed meat which is under refrigerator conditions [24]. Although L. monocytogenes will continue to thrive at low temperature as 1.1 °C [25] the storage temperature and duration of refrigerated storage before consumption are important factors which reduce the risks of foodborne listeriosis [26]. The recommended refrigerator temperature is 40 °F (4.4 °C); however, abuse home refrigerator temperature can rise to more than 12 °C [26,27].

Previous published studies indicated that the inhibitory efficacies of plant-derived antimicrobials may be affected by temperature [28,29]. The results from Exp. 1 showed that HEs 2, 4, 5 and 8 exhibited inhibitory effects against each of five L. monocytogenes strains at 37 °C. However, in order to use those four HEs as food preservatives, they must be effective against L. monocytogenes under food storage conditions. In this experiment, inhibition efficacies of those four HEs were evaluated at 12 °C which represented the abused refrigerator temperature. As discussed above, at concentrations of 1.56 and 0.78 mg/mL, HEs 2, 4, 5 and 8 inhibited growth of a five-strain L. monocytogenes cocktail at abuse refrigeration temperature of 12 °C, except herb extract 8 at the concentration of 0.78 mg/mL. At a concentration of 3.13 mg/mL, these four HEs reduced cell populations in a range of 2.2 to 1.6 log CFU/mL at 11 days. In a previous study, thyme essential oil showed the inhibitory effect against L. monocytogenes cocktails at 10 °C up to 12 days in minced beef [30]. HEs 2, 4, 5 and 8 were also expected to be developed into food preservatives for inhibiting and/or reducing foodborne L. monocytogenes. For example, those four HEs could be added to RTE foods as supplements or incorporated into food packaging materials to control L. monocytogenes growth. Further experiments should be conducted to determine the inhibitory effects and reductions of those four HEs in food products. In addition, since HEs carry specific odor, palatability of the food applied with HEs should be evaluated by sensory panel.

Conclusion

In summary, HEs 2, 4, 5 and 8 exhibited inhibitory effects against L. monocytogenes at 37 °C in a range of MIC between 5 - 50 mg/mL. HE 4 reduced cell populations of each selected strain ranged between 0.38 -

0.91 log CFU/mL after 30 min treatment at 37 °C. In addition, at concentrations of 1.56 and 0.78 mg/mL, HEs 2, 4, 5 and 8 inhibited growth of a five-strain L. monocytogenes cocktail at 12 °C, except the HE 8 at the concentration of 0.78 mg/mL. At a concentration of 3.13 mg/mL, these four HEs reduced cell populations in a range of 2.2 to 1.6 log CFU/mL at 11 days. For their practical application, further experiments should be conducted to determine the inhibitory effects and reductions of those HEs in a variety of food products. In addition, palatability of the foods which applied with HEs should be evaluated by sensory panel

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Iris Publishers- Open access Journal of Engineering Sciences | Biomimetics - A Potential Solution to Drag Reduction in Modern Aerodynamics

 

Authored by D Bhatia*,

Introduction

By the year 2050, global aviation emissions are expected to grow by 300-500% as compared to 2005 [1]. With climate change becoming a reality, there is increasing environmental, political, and economic pressure to improve fuel efficiency and reduce carbon emissions associated with transportation. The European Commission has set an ambitious target of reducing aviation emissions by 75% by 2050 [2].

Reduction in aircraft emissions can be brought either through improvements in propulsion technology or reduction in drag generated by the aircraft. In terms of propulsion technology, current trends tend to be geared towards electrification of the propulsion system as observed in the More Electric Aircraft (MEA) initiative [3]. However, commercial electric aviation is in its nascency and a mature technology, by conservative estimates, is 20-40 years away from fruition [4]. In the present scenario, drag reduction is one of the most promising methods to try and achieve this goal. It is estimated that even a 10% reduction in drag (through skin friction reduction) can lead to fuel savings of $200 million/year for the aviation sector [5]. Skin friction constitutes about half of all drag generated by the aircraft as shown in Figure 1 [6]. Reduction of friction drag would greatly contribute towards the reduction in emissions from aircraft. However, conventional drag reduction methods have either plateaued or possess great difficulty in their practical implementation-details of which can be found based on an extensive study by Jahanmiri [7].

The reduction in skin friction drag can be attained by looking at possible solutions from nature i.e. Biomimetics. Biomimetics, by definition, is the approach to human innovation by emulating nature. One only has to look at the relative ease at which sharks swim at high speed (75 kph), even though, they possess such a large mass (545 kg) to realize the low drag that they produce [8]. The low drag phenomenon of sharks can be explained by observing the shark skin. The shark skin (and those of other elasmobranch fishes such as skates, rays, etc.) are covered by minute placoid scales known as dermal denticles [9]. Sharks also possess very fine longitudinal ridges called riblets [9]. The dermal denticles and the riblets are also supplemented by a mucous layer which not only enable sharks to swim with minimum drag and catch prey but also promote anti-fouling behaviour [10-11].

Shark-skin inspired studies have been conducted extensively in the field of hydrodynamics with great efficacy. Biomimetic incorporation of the riblet based geometries has been the inspiration for drag reduction in the world of hydrodynamics since the last 30 years. Dean and Bhushan have presented a comprehensive review of riblet based drag reduction mechanisms in a hydrodynamic environment [12]. Biomimicry of these shark skin riblet based geometries became so popular that they were used in commercial applications such as the Speedo FSII swimsuit which demonstrated a drag reduction of 7.7% under live swimming conditions and 10-15% reduction under stiff-body conditions as compared to conventional swimsuits [13]. Thus, the shark-skin inspired riblets have shown great promise when used in water.

The authors believe that the biomimicry of the shark skin can also be translated to the field of aerodynamics. The authors base their opinion not only on existing data available from the hydrodynamic environment but also based on existing studies that make use of generic riblet shaped geometries to reduce turbulent skin friction drag. In most of these studies, the presence of a riblet shaped geometry either led to a net reduction in skin friction drag or led to a reduction in cross flow motion within the boundary layer and thereby reducing momentum transport away from the boundary layer [14-15].

However, there exist potential hurdles in the development of a riblet based features to enable drag reduction within the laminar boundary layer. Firstly, the positioning of these riblets need to be optimized within the laminar boundary layer (as opposed to the turbulent boundary layer in existing studies). Secondly, the size and shape of these riblets would have to be optimized based on the shark skin. The authors are of the opinion that on the completion of the optimization of these shark skin inspired riblet based geometries, these riblets could be placed strategically on the surface of the aircraft wing to try and reduce drag.

Biomimetics could also find its way into renewable energy applications such as wind turbines. Surface coatings bearing the same consistency as the mucous secreted on the shark skin could be used for anti-icing of wind turbine blades. Ice-accretion could be reduced on the surface owing to the use of the very low friction surface coating. The use of these biomimetic surface coatings could also contribute to self-cleaning wind turbine blades which would reduce debris accumulation and ice accretion on the surface of the turbine blades. A reduction in ice-accretion would reduce the drag on the wind turbine blades thereby improving their efficiency and reducing their downtime.

Thus, biomimetics provides us with a unique way forward to enable drag reduction without the use of complicated solutions. As Ockam’s Razor states, “The simplest solution is most likely the right one”, biomimetics gives us an opportunity to attain drag reduction using simple and straightforward solutions inspired by verifiable examples observed in nature.

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Iris Publishers-Open access Journal of Orthopedics Research | How to Halt Cancer

 

Authored by Andrew Hague*,

Abstract

The rules for the procedure to stop cancer are the same as those for repairing a puncture of a bicycle tyre. To make it easy for everyone to understand, this article will take you step by step through repairing a puncture and reversing cancer. The procedures are entirely different, but the rules are the same.

There is a problem

When the tyre is flat, the problem is obvious. When a person has a pain, it may be cancer, or it may be something else. You have to find out what has happened and that requires the right tools. To get the tyre off the rim to get to the inner tube, you need tyre levers, not spoons, screw drivers or your fingers. This is the first rule:

Get the right tool for the job

CellSonic has an easy way to diagnose cancer and every user is told about this when they buy a machine. To CellSonic, all cancers are the same. They all have a low voltage. It does not matter where they are in the body. The CellSonic pulses penetrate. Oncologists use less effective ways to diagnose cancer such as a circulating tumour cell test which analyses the blood but does not show where the tumour is, just that it is somewhere in the body. Scans produce pictures which locate tumours but only when the cluster of cells is big enough to show in a picture.

The CellSonic clinic checks a patient from head to toe regardless of where the patient says they have cancer. There are always more places to which the cancer cells have migrated, and all places have to be found and stopped. The same applies to the inner tube. Check if there are more holes, not just the first one you found. If you are in the workshop, get a basin of water and immerse the inflated inner tube to see where bubbles of air are emerging. If you are out on the road, maybe there is a puddle or a stream into which you can dunk the inner tube. It always helps to have the right tool for the job. Therefore, the right tool for cancer is a CellSonic VIPP machine. VIPP means very intense pressure pulse. It emits three forces: pressure, light and an electrical field like a hand-held thunderstorm.

Best to remove the wheel from the bicycle. Lifting the tyre off the bicycle rim is easy with tyre levers and almost impossible without. Be careful with the valve and take out the inner tube. Now find the leak. Pump the tyre up and listen for any hissing to reveal the hole or hold it against your cheek to feel the blow of air. Having found the hole, clean it with fine emery cloth and apply rubber solution. This is the second rule:

Everything has to work 100% every time

If the rubber solution does not work, the patch will not stick. If what you are going to do for cancer does not work, the cancer will not be stopped. Most oncologists will use chemotherapy. It has a success rate of 2.5%. Ask the doctor and if they don’t know, insist that they find out. A failure rate of 93% is total failure. If the rubber solution failed, the puncture will never be repaired. You don’t have enough patches or the time to be stranded at the roadside. Using chemotherapy is as stupid as trying to repair a puncture with oil.

If the bicycle chain squeaks, apply oil, essential on machinery and worse than useless on a puncture; it can rot the tyre and can be fatal on the rims to stop the breaks squealing. Adjust the brake blocks. Likewise, chemotherapy has many side effects, most cancer patients say it is worse than the cancer itself. Ask the doctor why they use chemotherapy, and they will say that it is standard procedure. Persist in questioning and you will learn about the biggest scam in history. Equally as bad is nuclear radiation used to target cancer cells but usually misses and causes cancer.

CellSonic aimed at the cancer cells stops the cancer being cancer during the treatment. The sensor is held in place and when it indicates that the voltage has risen to 75 millivolts, the cells are benign. We have had a case where a thyroid tumour on the throat shifted from malignant to benign with 41 pulses of the CellSonic machine. Zapping at 4 a second, it took ten seconds to make the lady’s cancer benign. I spoke to her a few days ago and she had been for scans which reported all benign. They could see that there had been cancer and now it was all gone.

Compare that with chemo and radiation and there is no contest. The right tool for the job that works every time. No one leaves a CellSonic clinic with cancer. It is checked during the treatment.

Rule number three:

No side effects

The CellSonic technology has a lot in common with lithotripsy, a technique of biophysics that has aimed very powerful pulses into millions of kidneys in many countries for forty years without side effects. This was the biggest safety study in medical history. With cancer, there absolutely must be no side effects. The patient is seriously weakened by their immune system struggling to eradicate mutant cells and it makes no sense to weaken them further with a lethal poison of chemotherapy. I cannot understand why this poison is used in medicine. It has no benefits. It is mustard gas, banned in warfare. Doctors inject it into patients with the lie that it is a cure for cancer. With a puncture, your hands will get dirty, and this is an unfortunate side effect. Some cyclists keep disposable gloves with their puncture outfit to protect their hands, but I never bother.

Leave the rubber solution to dry, at least five minutes. Never put a patch on when the solution is still wet. It was easier with natural rubber inner tubes on which the rubber solution worked without fail. Synthetic butyl tubes are tricky. Clean the area properly with emery cloth and let the solution dry thoroughly. Press the patch on firmly. You have repaired the puncture. Are you now ready to put the tube back in the tyre? No!

Rule number four.

Find the cause of the problem

Carefully run a finger around the inside of the tyre to feel for something that punctured the tube. It may well be a thorn still sticking through the tyre. Don’t let it cut your finger. These days, there is less broken glass in the road but if you ride on roads with hedges alongside, the hedge trimmers will scatter thorns in the road. If you see the debris, get off and walk. More about avoiding the problem comes later in this article.

CellSonic has stopped the cancer in the patient. Is the patient healed? Assume no. What caused the cancer? You have to find the cause in order to stop it coming back again. If you don’t find it, almost certainly it will still be there. How does cancer happen? It happens every day and is stopped every night. Our bodies are made of billions of cells that replicate on average every six weeks. New cells are formed as exact copies of the original cell. Statistically, with billions doing this during daytime, some will not be exact copies. They will be different and are called mutations. That is why we need an immune system so that we can repair ourselves automatically at night when we are asleep. Next morning, we awake clean and the process starts again. That is why it is essential to keep the immune system in good order.

A person with cancer will have had it for ten to fifteen years and only when the lumps, the tumours, have grown big enough to hurt does the person seek help. What happened years ago to prevent the immune system intercepting the cancer cells and killing them? That is what you have to find out. It is as essential as finding the thorn still protruding through the tyre. Just as putting the inner tube back in the tyre would have caused the tyre to go down within a mile, so will the ex-cancer patient succumb to cancer again if their immune system weakness is not found and helped.

They may have been chain smokers. Perhaps they worked with chemicals. Why were they addicted to tobacco? Did they not have protective clothing in the chemical factory? Did they think they were invincible? What caused the stress?

As I learn more about cancer, I find that in almost every case there was stress. The causes of stress are many. It is not the same as a challenge. Long distance cycling and daring mountaineering are challenges and in many ways the antidote to stress. Usually stress comes from relationships, people to people.

Does an oncologist open a conversation with the patient to see whether the stress persists? Hardly ever. If a trained oncologist learned anything about psychological trauma, they were not taught at medical school. They will have understood it from their own observations. Most of them would say that what happened years ago is none one their business. They have applied the standard treatment and if the patient has not responded at least they complied with the legally approved procedure and have no liability for the death of the patient which occurs in 97% of cases.

A CellSonic therapist will listen. The patient will always talk. They want to. They are opening their mind. If they will not, their immune system remains switched off. Many CellSonic machines are now being bought by ex-patients, people who have reversed their cancer. By their own experience they know what the machines can do. They have not sought medical papers or demanded random controlled clinical trials seeking to find the side effects. They know that CellSonic is utterly safe and want to help friends and relatives and can also see a way forward for themselves with a CellSonic clinic to give them independence. And there’s the clue. In many cases, a fresh start means leaving whoever caused them stress, doing good and being independent. Just as the bike tyre is ready to ride, so is the patient ready to face the world again safe in the knowledge that what attacked them years back is not going to hurt them ever again. Which gives us rule number five:

Avoid the Problem

Get off the bike and walk when the road is strewn with hedge cuttings. Some racing cyclists would run carrying the bike. A sensitive person will avoid toxic people. Interestingly, highly intelligent people tend to be sensitive. Introverts are more at risk than extroverts. They have to learn about themselves. If this means forming a new opinion of their parents and siblings, then so be it. The stresses their parents had are not wanted by their children. New relationships will form. No one is an island. Everybody relates to others. Be kind and others will be kind to you. The mind controls the body and controlling the mind is the way to health. The road ahead is clean, and you will not have a puncture (Figure 1).

The rules:
• Get the right tool for the job.
• Everything has to work 100% every time.
• No side effects.
• Find the cause of the problem.
• Avoid the problem.
Professor Hague has cycled seriously and regularly for over seventy years. He has repaired punctures in all conditions including one freezing, moonless night in the middle of the Atlas Mountains of Morocco with only the light of stars. He discovered how to reverse cancer in 2016 since when thousands of patients have been saved (Figure 2).

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Iris Publishers-Open access Journal of Civil & Structural Engineering | The Case of Strengthening the Base of a Deformed Retaining Wall

 

Authored by Sokolov NS*,

Abstract

The problem of increasing the bearing capacity of foundations is always an urgent problem in modern geotechnical construction. With additional increased external loads on existing restraint structures, the use of traditional technologies to ensure their stability is not always justified. Often there is an urgent need to use non-standard methods of strengthening the bases. There are frequent cases of using existing retaining reinforced concrete structures for new additional loads from newly erected objects. In such cases, the use of ERT drill piles and soil ERT anchors in most cases successfully solves many complex geotechnical problems of strengthening overloaded bases.

Keywords: Geotechnical construction; Electric discharge technology ERT; Drill injection pile ERT; Soil anchors ERT

Introduction

Ensuring the safe operation of newly erected facilities on sites with complex terrain and weak physical and mechanical characteristics requires a special approach [1-9]. Often, with new construction, you have to deal with previously strengthened slopes. Most often, they were reinforced with the help of recessed reinforced concrete structures used as retaining structures. In most cases, according to the design scheme, they belong to the building structures of the cantilever type. A distinctive feature of such walls from unfastened ones is that with even small additional external loads, they can be deforworldized. It is not necessary to say that they can perceive significant increased loads from a newly erected object. In such cases, a non-standard approach is needed in their use for the purpose of ensuring the sustainability of both the slope and the retaining wall itself. This article discusses the case of adaptation of the existing cantilever corner reinforced concrete retaining wall as a retaining reinforced concrete structure with significantly increased external loads of the roadbed under construction. It should be noted that the existing corner wall has already been deformed. The deviation from the vertical reached 950.0 mm with its height of 5.0 meters. It was decided to strengthen this wall with the help of additional drill-injection piles of ERT and turn from cantilevered to unfastened with the help of soil anchors ERT. Table 1 below shows one approach to using a deformed reinforced concrete retaining wall on a pile base of prismatic driven piles (Table 1).

Table 1: Resource requirements by component.

Figure 1 shows a plan of recessed reinforced with ERT drill piles, ground ERT anchors and monolithic reinforced concrete buttresses (Figure 1).
According to the results of engineering and geological surveys, the construction site is located in the south-eastern part of the Raduzhny residential micro district of the Moskov sky district of Cheboksary. Geomorphologically, the survey area occupies the left slope of the valley of the Cheboksarka River, in the sole and middle part of the slope dissected by a dense network of filled and buried ravines, with absolute elevations from 71.2 in the floodplain of the Cheboksarka River to 112-116.0 m in the north-western part of the survey site (the south-eastern part of the planned territory of the micro district. “Rainbow”). The height difference is about 45.0 m. The general slope of the territory is observed in the southern direction - towards the valley of the Cheboksarka River. The engineering and geological structure of the site to the studied depth (40.0 m) is represented by a thickness of bedrock of the Severodvinsk and Vyatka tiers of the Upper Permian department (P3s + v), overlapped from the surface with quaternary deposits of different ages and genesis. The entire thickness from above is covered with high-power bulk soils (tQIV). The hydrogeological conditions of the construction site to the studied depth (40.0 m) for the period of surveys in August 2018 are characterized by the presence of one non-pressure groundwater horizon. Groundwater is opened in all wells at depths of 0.2 - 23.8 m (abs.marks 71.1 - 100.6 m) and is confined to tQIV bulk soils, landslide deposits dpQ (P3s + v), alluvial deposits (aQIII), Upper Permian sands of shallow, dusty, medium size, water-saturated, sandy layers in Upper Permian clays and loams (aleurites) of sandy and calcareous marls (P3s+v). The water stop is the denser underlying Upper Permian clays (P3s+v). An engineering-geological section indicating the vertical binding of the existing and newly erected walls is shown in Figure 2 (Figure 2).

To use the existing retaining wall for the purpose of its perception of additional increased external loads, a project was developed for a device with the device of additional recessed reinforced concrete structures using ERT drill-injection piles, ground ERT anchors, monolithic reinforced concrete buttresses and the transformation of its design scheme from cantilevered to unfastened. Table 2 below shows the algorithm for the production of geotechnical works, divided into stages. It should be noted that their division into stages is associated with the need to ensure the stability of the slope during construction and to create safe working conditions (Table 2).

Table 2: Resource requirements by component Algorithm of geotechnical works.

Findings

1. The approach of adapting the existing restraint structure using ERT drill piles, ERT ground anchors and monolithic reinforced concrete buttresses considered in the article to create a completely new retaining fastening retaining wall.
2. The newly designed and erected retaining wall made it possible to ensure the stability of the overloaded base and created conditions for safe work.

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